STUDIES IN MYCOLOGY 50: 45–61. 2004. New ochratoxin A or sclerotium producing species in Aspergillus section Nigri 1* 1 1 2 Robert A. Samson , Jos A.M.P. Houbraken , Angelina F.A. Kuijpers , J. Mick Frank and Jens C. 3 Frisvad 1Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, the Netherlands; 233 Tor Road, Farnham, Surrey, GU9 7BY, U.K.; 3Center for Microbial Biotechnology, BioCentrum-DTU, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark *Correspondence: Robert A. Samson, [email protected] Abstract: Aspergillus section Nigri includes some of the most important species for biotechnology and its species are of widespread occurrence. During our surveys of various food products and tropical soil we isolated several aspergilli belonging to section Nigri. In this paper, four new sclerotium and/or ochratoxin A producing species belonging to this section are proposed. In addition, based on a polyphasic approach using traditional characters, extrolites and β-tubulin sequencing, a provisional revision and synoptic key of section Nigri is proposed. Aspergillus costaricaensis was isolated from soil in Costa Rica and produces large pink to greyish brown sclerotia. Aspergillus lacticoffeatus was found on coffee beans in Venezuela and Indonesia, and is an effective producer of ochratoxin A. Aspergillus piperis was isolated from black ground pepper and produces large yellow to pink brown sclerotia. Aspergillus sclerotioniger was isolated from a green coffee bean and produces large yellow to red brown sclerotia and abundant ochratoxin A. The species A. homomorphus is validated. The ochratoxin A producing black aspergilli are revised. Fifteen species are provisionally accepted in Aspergillus section Nigri, four of these produce ochratoxin A. Ochratoxin A producing species of section Nigri occurring on grapes, raisins and in wine include A. carbonarius and to a lesser extent A. niger. Four species recovered from coffee, viz. A. carbonarius, A. niger, A. lacticoffea- tus and A. sclerotioniger, all produce ochratoxin A, but other species of Nigri also occur on this substrate, including A. japonicus and A. tubingensis. The 10 species not producing ochratoxin A are especially interesting for biotechnological exploration, as many other extrolites are produced by these species. Taxonomic novelties: Aspergillus costaricaensis Samson & Frisvad sp. nov., Aspergillus homomorphus Frisvad & Samson sp. nov., Aspergillus lacticoffeatus Samson & Frisvad sp. nov., Aspergillus piperis Samson & Frisvad sp. nov., Aspergillus sclerotioniger Steiman, Guiraud, Sage & Seigle-Mur. ex Samson & Frisvad sp. nov. Key words: Aspergillus niger, black aspergilli, ochratoxin A, pyranonigrin, sclerotia. INTRODUCTION echinulate conidial ornamentations distinct from the remaining black Aspergillus taxa, which produce The black aspergilli are among the most common verrucose conidia. Within the verrucose category, A. fungi causing food spoilage and biodeterioration of fonsecaeus, A. acidus, A. niger var. niger, A. niger other materials. They have also been extensively used var. phoenicis, A. niger var. ficuum, A. niger var. for various biotechnological purposes, including tubingensis, A. niger var. pulverulentus, A. niger var. production of enzymes and organic acids (Schuster et awamori, A. citricus (A. foetidus) and A. citricus var. al. 2002). The taxonomy of Aspergillus section Nigri pallidus were recognized. has been studied by many taxonomists and was re- Aspergillus niger is the most frequently reported cently reviewed by Abarca et al. (2004). Mosseray species in this section, and has often been included in (1934) described 35 species black aspergilli, while biotechnological processes that are Generally Re- Raper and Fennell (1965) reduced the number of garded as Safe (GRAS). However, species concepts species accepted within their A. niger group to 12. Al- are uncertain in this complex and occasionally the Musallam (1980) revised the taxonomy of the A. niger name A. niger has been used for any member of the group, primarily based on morphological features. She section. Taxonomic studies using molecular methods recognized seven species (A. japonicus, A. carbon- have divided the A. niger complex into two species, A. arius, A. ellipticus, A. helicothrix, A. heteromorphus, niger and A. tubingensis (for overview see Abarca et A. foetidus, A. niger), and described A. niger as an al. 2004). Some further species have been described aggregate consisting of seven varieties and two for- but not considered in revisions or reviews. Aspergillus mae. Kozakiewicz (1989) distinguished A. ellipticus, ellipsoideus was described as a new species with A. heteromorphus, A. japonicus, A. helicothrix, A. ellipsoidal greyish black conidia (Rai & Chowdhery atroviolaceus and A. carbonarius as species exhibiting 1979). Aspergillus homomorphus and A. pseudohet- 45 SAMSON ET AL. eromorphus were invalidly described (no designated The names of colours are based on Kornerup & Wan- type, International Code of Botanical Nomenclature scher (1978). The cultures used for the molecular Art. 37) (Steiman et al. 1994; see Mouchacca 1999). study were grown in 2 mL malt peptone (MP) broth Recently, a new species A. vadensis, with a different using 10 % (v/v) of malt extract (Brix 10) and 0.1 % extrolite profile, colony characters and unusually low (w/v) bacto peptone (Difco) in 15 mL polystyrene citric acid production, was proposed (de Vries et al. centrifuge tubes. The cultures were incubated at 25 °C 2004a, b). without agitation for 7 d in light/darkness. The strains Ueno et al. (1991) were the first to report on ochra- selected included 1 to 8 representatives of the major toxin A (OA) production by a black Aspergillus taxa accepted by Al-Musallam (1980), Kozakiewicz species, A. foetidus. This was later confirmed by (1989) and Abarca et al. (2004) (see Table 1) in Téren et al. (1996) and Magnoli et al. (2003). Abarca addition to the new taxa described here and in de et al. (1994) reported that two strains of A. niger Vries et al. (2004b). produced OA, which was confirmed in numerous studies (Ono et al. 1995, Téren et al. 1996, 1997, Extrolite analysis Nakajima et al, 1997, Heenan et al. 1998, Accensi et Extrolites (includes secondary metabolites; for defini- al. 2001, Urbano et al. 2001, Dalcero et al. 2002, Da tion see Samson & Frisvad 2004) were analysed by Rocha et al. 2002, Abarca et al. 2003, Magnoli et al. HPLC using alkylphenone retention indices and diode 2003, Taniwaki et al. 2003, Suárez-Quiroz et al. array UV-VIS detection as described by Frisvad & 2004). Horie (1995) reported OA in A. carbonarius, Thrane (1987), with minor modifications as described which was confirmed by Wicklow et al. (1996), Téren by Smedsgaard (1997). Standards of ochratoxin A and et al. (1996), Heenan et al. (1998), Varga et al. B, aflavinine, asperazine, austdiol, kotanin and other (2000), Joosten et al. (2001), Da Rocha et al. (2002), extrolites from the collection at Biocentrum-DTU Cabanes et al. (2002), Sage et al. (2002), Abarca et al. were used to compare with the extrolites from the (2003), Battilani et al. (2003), Taniwaki et al. (2003), species under study. Pyranonigrin A was identified by Bellí et al. (2004) and Sage et al. (2004). Varga et al. comparison with literature UV and MS data (Hiort (2000) tested about 160 black Aspergillus strains from 2003, Hiort et al. 2004) collections and from field isolates for OA production using an immunochemical method and thin layer DNA Extraction, sequencing and analysis chromatography. The strains examined included 12 A. The total fungal genomic DNA was isolated using carbonarius and 45 A. japonicus strains from culture UltracleanTM Microbial DNA Isolation Kit (MoBio, collections and field isolates from all over the world, Solana Beach, U.S.A.) according to the manufac- including about 100 strains belonging to the A. niger turer’s instructions. Amplification of β-tubulin gene species complex. was mostly performed using the primers Bt2a and Ochratoxin A production was detected in about 6 Bt2b. Some strains in this study Bt-T10 (5'ACG ATA % of the strains from the A. niger species complex GGT TCA CCT CCA GAC 3') an Bt2b (Glass 1995). (Abarca et al. 1994, Téren et al. 1996). Of the 13 A. PCR was performed in a 25 µL reaction mixture carbonarius strains tested, six produced both OA and containing 1 µL of genomic DNA (10 ng/µL), 0.75 µL ochratoxins B (Fig. 8, Téren et al. 1996, Wicklow et of MgCl2 (50mM provided with BioTaq), 2.5 µL al. 1996). Aspergillus ellipticus, A. heteromorphus, A. Buffer with 10× NH4 (provided with BioTaq), 17.8 µL japonicus and A. tubingensis strains did not produce of ultra pure sterile water, 1.85 µL dNTP (1 mM), detectable amounts of ochratoxins. However, A. 0.50 µL of each primer (10 pmol/µL) and 0.1 µL japonicus was later claimed to produce OA (Dalcero BioTaq polymerase (5 U/µL, BiotaqTM DNA Poly- et al. 2002, Battilani et al. 2003). merase, Bioline Randolph, U.S.A.). Amplification was During our surveys of coffee, black pepper and performed in a GeneAmp PCR system 9700 (AB soil, several isolates of black aspergilli were recov- Applied Biosystems, Nieuwerkerk a/d IJssel, The ered. The purpose of this paper is to describe four new Netherlands); programmed for 5 min 94 °C followed species from section Nigri, distinguished from previ- by 35 cycles of 1 min denaturation at 94 °C followed ously known species by large sclerotia or unusual by primer annealing 1 at 55 °C and primer extension 1 conidial colours. Furthermore we wanted to suggest a min. at 72 °C and a final 7 min elongation step at 72 provisional revision of this industrially important °C. After amplification of the β-tubulin gene, excess section of Aspergillus based on a relatively small primers and dNTP’s were removed from the reaction number of typical strains of each taxon. mixture using a commercial GFX column, PCR DNA Purification kit (Amersham Bioscience, Roosendaal, MATERIALS AND METHODS The Netherlands). The purified PCR fragments were resuspended in 50 µL of TE buffer.